Position information acquisition device and position information acquisition method for biological sample in paraffin block

Abstract

To solve the problem that there is a risk of wasting a glass substrate by placing a thin sliced piece not including a biological sample on the glass substrate since there is no method for three dimensionally obtaining the position of the paraffin-embedded biological sample in a thin sliced piece manufacturing process for manufacturing a pathological slide for performing pathological diagnosis.SOLUTION: To acquire three dimensional position information of a biological sample in a paraffin block which is effective in a slicing process by using an optical coherence tomography technique.SELECTED DRAWING: Figure 12

Description

[Technical Field]

[0001] Pathological diagnosis is a diagnosis performed by observing, under a microscope, pathological section slides prepared from sections taken from a patient for testing purposes or from a site removed by surgery, after fixation, sectioning, and staining processes, with the aim of determining the patient's disease. In recent years, with the emergence of therapeutic drugs called molecular targeted drugs that are suited to the type of disease a patient has, pathological diagnosis is no longer limited to diagnosing whether a tumor is benign or malignant, but is also being performed to determine the drug to be used for treatment.

[0002] As the number of opportunities for pathological diagnosis increases, the number of pathological section slides produced also increases, and the number of subsequent stainings also increases.The increase in the number of pathological section slides produced and stained also increases the burden on the work staff. Pathological section slides are made by fixing tissue taken from a patient with formalin solution or similar, embedding it in paraffin, cutting it into slices several microns thick using a thin-sectioning device, and placing it on a glass slide. Then, a prescribed staining process is carried out.

[0003] As shown in FIG. 1, a paraffin-embedded biological sample to be used in a thin-sectioning device is mounted on a plastic molded part 31, in which a fixed biological sample 11 is embedded (held) in paraffin 21. In the thin-sectioning device, the molded part 31 is fixed, and the paraffin block is cut away from the surface 21a of the paraffin 21. The paraffin block is cut away from the surface 21a of the paraffin 21. Once the blade reaches position 11a, which is close to the surface 21a of the paraffin 21 of the biological sample 11, the biological sample 11 is contained in the cut section, and thin sections suitable for pathological diagnosis can be produced. However, because the biological sample 11 is not contained in the cut sections produced until the blade reaches position 11a, which is close to the surface 21a of the paraffin 21 of the biological sample 11, it is desirable to bring the blade to position 11a as quickly as possible during the thin-sectioning process. Furthermore, the thin slices produced in the process up to the point where the blade reaches 11a, which is close to the surface 21a of the paraffin 21 of the biological sample 11, do not contain the biological sample 11, so if these thin slices are mounted on a glass substrate, the glass substrate will be wasted.

[0004] As shown in Figure 2, in a paraffin-embedded biological sample, the distance from the surface 21a of the paraffin 21 to a position 12a of the biological sample 12 embedded in the paraffin 21 that is close to the surface 21a of the paraffin 21 is not constant, and may be greater than the distance from 21a to 11a shown in Figure 1.

[0005] Furthermore, as shown in Figure 3, fragments 13d of the biological sample 13 may be present within the paraffin 21 between the surface 21a of the paraffin 21 and 13a, which is the position of the main surface of the biological sample 13 embedded in the paraffin 21 that is close to the surface 21a of the paraffin 21, although this position is below the position 13a, which is the position of the main surface of the biological sample 13 embedded in the paraffin 21 that is close to the surface 21a of the paraffin 21.

[0006] In the case shown in Figure 3, if a pathological diagnosis is performed using a thin section containing fragments 13d of the biological sample 13, the pathological diagnosis will be performed using a portion that is not a biological sample below the surface 13a that should be diagnosed, which is undesirable. Therefore, technicians with the skilled techniques for performing thin sectioning take measures such as using information from other biological blocks extracted from the same patient to avoid producing slides containing thin sections that contain fragments 13d of the biological sample 13.

[0007] Therefore, if three-dimensional information about the position of a paraffin-embedded biological sample can be obtained, even an unskilled technician can efficiently perform the thin-sectioning process.

[0008] Furthermore, there is an automatic thin-section production device for producing pathological section slides, as shown in Patent Document 1. However, this device does not reflect the thinking ability of a skilled technician, and therefore may cause the device to place a section that does not contain a biological sample on the glass slide, or to place a section of a portion that is not used for pathological diagnosis, such as a fragment of the biological sample, on the glass slide, resulting in a loss of material.

[0009] If three-dimensional information about the position of the paraffin-embedded biological specimen can be obtained, it is possible to prevent material loss due to the automatic thin-sectioning device mounting a section that does not contain a biological specimen onto a glass slide, or mounting a section of a biological specimen fragment or other part that is not used for pathological diagnosis onto a glass slide, and also to reduce the working time. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Patent No. 5017704 specification

[0011] [Non-Patent Document 1] Yoshihisa Kushida, Nobutake Ozeki, Mitsuru Mizuno, Hisako Katano, Koji Otabe, Kunikazu Tsuji, Hideyuki Koga, Koichiro Kishima, Yoshio Soma, Ichiro Sekiya, "Two- and three-dimensional optical coherence tomography to differentiate degenerative changes in a rat meniscectomy model," Journal of Orthopaedic Research, Vol. 38, pp. 2592-2600 (2020) Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention has been made in consideration of the above points, and aims to obtain three-dimensional information on the position of paraffin-embedded biological samples, thereby shortening the work time for the thin section production process, preventing material loss, and enabling the process to be carried out efficiently. [Means for solving the problem]

[0013] To solve this problem, the device and method for acquiring positional information of a biological sample in a paraffin block of the present invention use optical coherence tomography to acquire three-dimensional positional information of the biological sample in the paraffin block, which is effective in the thin-sectioning process.Furthermore, the optical coherence tomography to acquire three-dimensional positional information of the biological sample in the paraffin block using the optical coherence tomography to acquire information acquired by an optical camera in addition to the three-dimensional positional information of the biological sample in the paraffin block, which is effective in the thin-sectioning process. [Effects of the Invention]

[0014] The positional information acquisition device for a biological sample in a paraffin block and the positional information acquisition method for a biological sample in a paraffin block of the present invention have the effect of shortening the working time of the thin section production process, preventing material loss, etc., and enabling the thin section production process to be carried out efficiently. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram of a paraffin block in which a biological sample is embedded. [Figure 2] FIG. 1 is a schematic diagram of a paraffin block in which a biological sample is embedded. [Figure 3] FIG. 1 is a schematic diagram of a paraffin block in which a biological sample is embedded. [Figure 4] FIG. 1 is a schematic diagram showing a method for fixing a paraffin block. [Figure 5] FIG. 1 is a schematic diagram showing a method for fixing a paraffin block. [Figure 6] 1 is a schematic diagram illustrating the configuration of a paraffin block optical coherence tomography system according to the present invention. [Figure 7] 1 is an example of data showing the position of a biological sample in a paraffin block acquired by optical coherence tomography. [Figure 8] This is an example of 3D data of the position of a biological sample in a paraffin block obtained by optical coherence tomography. [Figure 9] 1 is a schematic diagram illustrating the configuration of an optical photographing system for a paraffin block according to the present invention. [Figure 10] FIG. 1 is a diagram showing an example of three-dimensional position information output from the device for acquiring three-dimensional position information of a biological sample in a paraffin block according to the present invention. [Figure 11] FIG. 1 is a diagram showing an example of three-dimensional position information output from the device for acquiring three-dimensional position information of a biological sample in a paraffin block according to the present invention. [Figure 12] 1 is a schematic diagram illustrating the configuration of a paraffin block optical coherence tomography system according to the present invention. [Figure 13] 1 is a schematic diagram illustrating the configuration of an optical photographing system for a paraffin block according to the present invention. [Figure 14] 1 is a schematic diagram illustrating a mechanism for switching between a paraffin block optical coherence tomography imaging system and an optical imaging system according to the present invention. [Figure 15] FIG. 1 is a data acquisition flow diagram of the device for acquiring three-dimensional position information of a biological sample in a paraffin block according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] An apparatus for acquiring three-dimensional position information of a biological sample in a paraffin block as a first embodiment of the present invention will be described with reference to FIGS. 4 to 6. FIG. 4 is a cross-sectional view showing the mounting portion of the paraffin-embedded biological sample shown in FIG. 1. For fixation, a molded part 31, which is a plastic part on which the biological sample 11 is mounted while being embedded (held) in paraffin 21, is held by a holding member 41. As shown in the top view of FIG. 5, the holding member 41 has an uneven portion 42 formed thereon. The uneven portion 42 is, for example, a circular groove portion having a depth of 0.1 mm and a diameter of 1 mm formed on a surface 41a of the holding member 41.

[0017] FIG. 6 shows an optical coherence tomography system 1 that uses optical coherence tomography to measure a paraffin-embedded biological sample held by the holding member 41 shown in FIGS. 4 and 5 . Optical coherence tomography, also used in Non-Patent Document 1, is a method capable of obtaining refractive index information in the depth direction at the position where light is irradiated. The position where light is irradiated is manipulated in two dimensions to measure the three-dimensional structure of the object being measured in the manipulated area. In the optical coherence tomography of the present invention, for example, a system with a central wavelength of 1064 nm is used to measure a paraffin block having a square shape with sides of 20 mm at lattice positions spaced at 50 μm intervals, and a holding member 41 including the uneven portion 42 at lattice positions spaced at 100 μm intervals. Therefore, the optical coherence tomography unit 51 is equipped with a galvanometer mirror or a movable mirror with a function similar to a galvanometer mirror that moves the measurement light 50 to lattice positions spaced at 50 μm or 100 μm intervals. The optical coherence tomography unit 51 is connected to a computer device 61 that performs control and calculations of measurement results. In the optical coherence tomography system 1, measurement is performed with the holding member 41 fixed in contact with the sample holding section 53, thereby preventing the biological sample from moving during measurement.

[0018] 7 shows an example of the results of measuring a paraffin-embedded biological sample held by the holding member 41 shown in FIGS. 4 and 5 by optical coherence tomography. The results shown in FIG. 7 are obtained by performing optical coherence tomography along the line 99 shown in FIG. 5, and show information in the depth direction at the position on the line 99, with the left side of FIG. 7 corresponding to the upper side of the line 99 in FIG. 7. In FIG. 7, the surface 41a of the holding member 41, the surface 31a of the molded part 31, The height relationship of the surface 21a of the paraffin 21 is obtained. Here, because the paraffin 21 is not an opaque material, positional information of the surface 11a of the biological sample 11 embedded in the paraffin 21 is also obtained. Depth information measured by optical coherence tomography takes into account the refractive index of the medium. Therefore, the positional information of the surface 11a of the biological sample 11 is multiplied by approximately 1.45, which is the refractive index of the paraffin material. In the results shown in Figure 7, if the distance from the position of the surface 21a of the paraffin 21 to the position of the surface 11a of the biological sample 11 is obtained as 2.9 mm, this 2.9 mm is the actual distance multiplied by approximately 1.45, which is the refractive index of the paraffin material. Therefore, the distance from the position of the surface 21a of the paraffin 21 to the position of the surface 11a of the biological sample 11 is 2 mm, which is the value obtained by dividing 2.9 by 1.45.

[0019] Figure 7 shows the depth information obtained when optical coherence tomography measurements were performed along the position of line 99 shown in Figure 5. However, by performing optical coherence tomography measurements in two dimensions, it is possible to obtain three-dimensional information on the positional information of the surface 11a of the biological sample 11, i.e., height information in the area where the measurement was performed.

[0020] In the present invention, since it is desirable to acquire height information of the surface 11a of the biological sample 11 in the thin-section production process, the output information was height information from the surface 41a of the holding member 41. The height information of the surface 11a of the biological sample 11 from the surface 41a of the holding member 41 can be calculated by first calculating height information of the surface 21a of the paraffin 21 from the surface 41a of the holding member 41, and then calculating the distance between the surface 21a of the paraffin 21 and the surface 11a of the biological sample 11. As described above, the height information acquired by optical coherence tomography is a numerical value that takes into account information about the refractive index of the medium. Therefore, when measurements are performed using the measurement system shown in FIG. 6 , the height information of the surface 21a of the paraffin 21 from the surface 41a of the holding member 41 can be calculated using the same numerical value for the height difference because the medium is air. However, when calculating the distance between the surface 21a of the paraffin 21 and the surface 11a of the biological sample 11, the measurement result needs to be divided by 1.45, which is the refractive index of paraffin.

[0021] 8 shows an example of three-dimensional information of the position of the surface 11a of the biological sample 11 from the surface 41a of the holding member 41. In this data, the position of the surface 31a of the molded part 31 is displayed at positions where the biological sample 11 is not present, and is represented by a numerical value of -5. The position where the biological sample 11 is present is indicated by an outline 121, and the position of the surface 11a of the biological sample 11 is represented by a positive numerical value that is approximately 3.4 mm to 3.5 mm higher than the surface 41a of the holding member 41.

[0022] In this data example, the slicing position of the biological sample 11 in the thin section production process is 3.6 mm higher than the surface 41a of the holding member 41, and therefore the biological sample 11 does not exist in the paraffin 21, so there is no need to mount the sliced ​​sample on a glass slide. Also, if there is room for the size of the sample, it is sufficient to prepare a glass slide once the sample reaches a position 3.4 mm higher than the surface 41a of the holding member 41. In this way, if the three-dimensional position information of the biological sample 11 in the paraffin block is available before the thin section production process, it is possible to easily improve the efficiency of the work and reduce the waste of materials.

[0023] 9 shows an optical imaging system 3 that uses an optical camera to acquire the position of the biological sample 11 in the paraffin 21 held in the holding member 41 shown in FIGS. 4 and 5. A lens 72 is fixed to the optical camera 71, and the system captures images together with the alignment marks 42 formed on the surface 41a of the holding member 41. The camera 71 is controlled by a connected computer device 61, and the captured images are also stored and processed in the computer device 61.

[0024] Figure 10 shows data presented to the user after measurement of a biological sample in a paraffin block by optical coherence tomography using the optical coherence tomography unit 1 shown in Figure 6 and imaging using the optical imaging system 3 shown in Figure 9. The user can easily understand the data acquired by optical coherence tomography by viewing the image of the paraffin block taken by the camera shown in Figure 10(a) and the three-dimensional measurement results obtained by optical coherence tomography shown in Figure 8 (an example of which is shown in Figure 10(b)).

[0025] Although depth information of the biological sample cannot be obtained from the image of the paraffin block captured by the camera, information on the shape of the biological sample can be obtained. The shape of the biological sample can be superimposed on the captured image as the outline 111 of the sample.

[0026] In measurements using optical coherence tomography, a line 121 indicating the shape of the biological sample can be obtained from height information of the biological sample, as explained in Fig. 8. In Fig. 10, the line 111 indicating the shape of the sample is superimposed on the image of the paraffin block taken by the camera shown in Fig. 10(a), and the line 121 indicating the shape of the biological sample is superimposed on the three-dimensional information obtained by optical coherence tomography shown in Fig. 10(b), making it easier to understand the data obtained by optical coherence tomography. Here, when generating FIGS. 10(a) and 10(b), it is desirable to use the uneven portion 42 on the holding member 41 as a positioning mark and align the position information of the image from the position information.

[0027] For users, making it easier to understand data acquired by optical coherence tomography also has the advantage of making it easier to notice when an error occurs in the device.

[0028] FIG. 11 shows an example of a case where the biological sample in paraffin has irregularities. FIG. 11(b) shows the results of optical coherence tomography, in which outlines 123 indicating the shape of the irregularities obtained by optical coherence tomography are shown together with outlines 122 indicating the shape of the biological sample. FIG. 11(a) shows image data in which outlines 112 of the sample have been added to an image taken by the optical camera shown. This makes it easier for users to understand the data obtained by optical coherence tomography, even when the biological sample has irregularities.

[0029] In the position information acquisition device and position information acquisition method of the present invention for a biological sample in a paraffin block, height information of the biological sample is information acquired by optical coherence tomography, but by providing an imaging system using an optical camera and combining this with information acquired by the optical camera and providing it to the user, the user's understanding of the data is enhanced and it is possible to respond more quickly if an error occurs in the device.

[0030] FIG. 12 illustrates an optical coherence tomography system 2 for measuring a paraffin-embedded biological sample using optical coherence tomography as a second embodiment of the present invention. The optical coherence tomography system 2 shown in FIG. 12 is configured such that the sample portion of the optical coherence tomography system 1 shown in FIG. 6 is placed in a water tank 81 and immersed in water 80. The optical coherence tomography system 1 shown in FIG. 6 is also configured to be tilted overall. As described in Non-Patent Document 1, in optical coherence tomography, placing the sample underwater prevents the outermost edge from being too bright and allows for the detection of subtle differences in refractive index within the sample. This method makes it possible to obtain refractive index information in the depth direction at the position where light is irradiated. Furthermore, by ensuring that the laser light 50 for optical coherence tomography is not perpendicular to the water surface 80a, the reflected image from the water surface is prevented from adversely affecting the data of the object being measured.

[0031] 13 shows an optical imaging system 4 that acquires the position of a biological sample 11 in paraffin 21 using an optical camera 71. The optical imaging system 4 has an inclined configuration similar to the optical coherence tomography system 2. When the optical imaging system 4 is placed at the imaging position, the level of the water 80 in the water tank 81 is lowered so that the sample exists in the air rather than in the water. Therefore, the water tank 81 is provided with a drain valve 83 and a water supply valve 82 for reducing the amount of water 80 in the water tank.

[0032] Since it is desirable that the optical coherence tomography imaging unit 51, optical camera 71, and lens 72 are positioned parallel to the paraffin 21, the optical coherence tomography imaging unit 51, optical camera 71, and lens 72 are an integrated structure 90 arranged on a single base 91 as shown in Figure 14, and it is desirable that by moving the base 91, the optical system performing optical coherence tomography imaging shown in Figure 12 and the image capturing optical system shown in Figure 13 move in conjunction with each other, and be quickly positioned at the sample capturing position.

[0033] FIG. 15 shows a flow 100 for quickly performing optical coherence tomography of the paraffin 21 using the optical coherence tomography system 2 and optical imaging of the paraffin 21 using the optical imaging system 4.

[0034] Even if water droplets adhere to the paraffin 21, this does not cause a problem in the process of producing thin slices. Therefore, it is preferable to perform the process of optically photographing the paraffin 21 using the optical imaging system 4 before performing optical coherence tomography of the paraffin 21 obtained using the optical coherence tomography system 2, as this eliminates the need to carefully remove water droplets adhering to the surface of the paraffin 21 after the optical coherence tomography.

[0035] 15, after the sample is set, the optical imaging system 4 is placed in a predetermined position, and imaging is performed by the optical camera 71. Then, the water supply valve 82 of the water tank 81 is activated, and the water surface 80a rises to a position that covers the paraffin 21 and the holding member 41. The optical coherence tomography system 2 then moves to a position where optical coherence tomography is performed, and optical coherence tomography is performed. Then, the drain valve 83 is activated, the water level is lowered, and the sample is removed while data is output.

[0036] In the process flow of the present invention, as described above, optical imaging of the paraffin 21 using the optical imaging system 4 is performed prior to the optical coherence tomography imaging process, thereby enabling rapid data acquisition. [Industrial Applicability]

[0037] The device for acquiring positional information of a biological sample in paraffin and the method for acquiring positional information of a biological sample in paraffin of the present invention have the effect of shortening the working time of the thin section production process, preventing material loss, etc., and enabling the thin section production process to be carried out efficiently, thereby contributing to cost reduction in medical institutions that perform pathological diagnoses. [Explanation of symbols]

[0038] 1, 2...optical coherence tomography system, 3, 4...optical imaging system, 11, 12, 13...biological sample, 21...paraffin, 31...mold part, 41...holding member, 42...concave and convex part, 50...optical coherence tomography laser light, 51...optical coherence tomography unit, 53...sample holder, 61...computer device, 71...optical camera, 72...lens, 80...water, 81...water tank, 82...water supply valve, 83...drain valve, 90...integrated structure, 91...base, 100...process flow, 111, 112, 121, 122, 123...outline of sample

Claims

1. a sample mounting section for mounting a block made mainly of paraffin in which a biological sample is enclosed; an optical coherence tomography unit that measures a paraffin block placed on a sample mounting unit; A device for acquiring positional information of a biological sample in a paraffin block, characterized by outputting positional information of the biological sample based on the position of the component on which the block is mounted or the component holding the component on which the block is mounted.

2. 2. The apparatus for acquiring position information of a biological sample in a paraffin block according to claim 1, wherein optical coherence tomography is performed underwater.

3. 3. The positional information acquisition device for a biological sample in a paraffin block according to claim 2, characterized in that in the above-mentioned device for acquiring positional information of a biological sample in a paraffin block, optical coherence tomography is performed underwater, and the optical axis of the optical coherence tomography and the main surface of the block are inclined with respect to the direction of gravity so that the measurement light for the optical coherence tomography is not perpendicular to the surface of the water covering the biological sample.

4. a sample mounting section for mounting a block made mainly of paraffin in which a biological sample is enclosed; a camera optical system for photographing the block; an optical coherence tomography unit that measures a paraffin block placed on a sample mounting unit; A device for acquiring positional information of a biological sample in a paraffin block, characterized by outputting positional information of the biological sample based on the position of the component on which the block is mounted or the component holding the component on which the block is mounted.

5. 5. The apparatus for acquiring position information of a biological sample in a paraffin block according to claim 4, wherein optical coherence tomography is performed underwater.

6. In the positional information acquisition device for a biological sample in a paraffin block, the optical coherence tomography is performed underwater, and the optical axis of the optical coherence tomography and the main surface of the block are inclined with respect to the direction of gravity so that the measurement light for the optical coherence tomography is not perpendicular to the surface of the water covering the biological sample.

7. a sample mounting section for mounting a block made mainly of paraffin in which a biological sample is enclosed; an optical coherence tomography unit that measures a paraffin block placed on a sample mounting unit; A method for acquiring positional information of a biological sample in a paraffin block, characterized by outputting positional information of the biological sample based on the position of the component on which the block is mounted or the component holding the component on which the block is mounted.

8. 8. The method for obtaining position information of a biological sample in a paraffin block according to claim 7, wherein the optical coherence tomography is performed underwater.

9. 9. The method for obtaining positional information of a biological sample in a paraffin block according to claim 8, characterized in that the optical coherence tomography is performed underwater, and the optical axis of the optical coherence tomography and the main surface of the block are inclined relative to the direction of gravity so that the measurement light for the optical coherence tomography is not perpendicular to the surface of the water covering the biological sample.

10. A method for obtaining positional information of a biological sample in a paraffin block, characterized by measuring a block composed primarily of paraffin in which a biological sample is encapsulated using optical coherence tomography, capturing image information using an optical camera, and outputting positional information of the biological sample from the surface of the component on which the block is mounted or the component that holds the component on which the block is mounted.

11. The method for obtaining position information of a biological sample in a paraffin block according to claim 10, wherein the optical coherence tomography is performed underwater.

12. 12. The method for obtaining positional information of a biological sample in a paraffin block according to claim 11, characterized in that, in the method for obtaining positional information of a biological sample in the paraffin block, optical coherence tomography is performed underwater, and the optical axis of the optical coherence tomography and the main surface of the block are inclined with respect to the direction of gravity so that the measurement light for the optical coherence tomography is not perpendicular to the surface of the water covering the biological sample.

13. The method for obtaining positional information of a biological sample in a paraffin block, as described in claim 11, characterized in that, in the method for obtaining positional information of a biological sample in the paraffin block, photography is performed using an optical camera before optical coherence tomography.

Images